FUNCTIONAL MAPPING OF THE SURFACE RESIDUES OF HUMAN THROMBIN

Utilizing site directed mutagenesis, 77 charged and polar residues tha t are highly exposed on the surface of human thrombin were systematica lly substituted with alanine. Functional assays using thrombin mutants identified residues that were required for the recognition and cleava ge of the procoagulant substrate fibrinogen (Lys(21), Trp(50), Lys(52) , Asn(53)+Thr(55), Lys(65), His(66), Arg(68), Tyr(71), Arg(73), Lys(77 ), Lys(106)+Lys(107), Asp(193)+Lys(196), Glu(202), Glu(229), Arg(233), Asp(234)) and the anticoagulant substrate protein C (Lys(21), Trp(50) , Lys(65), His(66), Arg(68), Tyr(71), Arg(73), Lys(77), Lys(106)+Lys(1 07), Glu(229), Arg(233)), interactions with the cofactor thrombomoduli n (Gln(24), Arg(70)) and inhibition by the thrombin aptamer, an oligon ucleotide-based thrombin inhibitor (Lys(65) , His(66), Arg(7)0, Tyr(71 ) Arg(73)). Although there is considerable overlap between the functio nal epitopes, distinct and specific residues with unique functions wer e identified. When the functional residues were mapped on the surface of thrombin, they were located on a single hemisphere of thrombin that included both the active site cleft and the highly basic exosite 1. N o functional residues were located on the opposite face of thrombin. R esidues with procoagulant or anticoagulant functions were not spatiall y separated but interdigitated with residues of opposite or shared fun ction. Thus thrombin utilizes the same general surface for substrate r ecognition regardless of substrate function although the critical cont act residues may vary.